PROKARYOTIC EXPRESSION OF RECOMBINANT MOUSE OP-1 AND ITS OSTEOGENESIS IN VITRO

2002 ◽  
Vol 06 (01) ◽  
pp. 9-15
Author(s):  
Zhenhua Lu ◽  
T. Sam Lindholm
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Development Research ◽  
Activity Assay ◽  
Rt Pcr ◽  
Iptg Induction ◽  
Reading Frame ◽  
Prokaryotic Expression Vector ◽  
Osteogenesis In Vitro

OP-1 is a main member of the BMP family. It plays diverse and significant roles in growth and differentiation. RT-PCR amplified the open reading frame of mouse OP-1 from cDNAs synthesis from CD-1 mouse embryo. By coding the gene of mOP-1 was inserted into pTrcHis 2B, the prokaryotic expression vector through IPTG induction, and the recombinant protein was isolated and purified with Ni-NTA resin. In an ALP activity assay, rmOP-1 has shown osteogenesis activity in vitro. It would offer a fast and inexpensive means of the productions of recombinant protein and an easy tool for growth and development research on rmOP-1.

Cloning and expression ofSpodoptera lituranucleopolyhedrovirusgp41gene inEscherichia coliand preparation of antibody

2005 ◽  
Vol 2 (2) ◽  
pp. 113-117
Author(s):  
Pan Li-Jing ◽  
Li Zhao-Fei ◽  
Yin Chong ◽  
Lv Lei ◽  
Pang Yi
Keyword(s):  
Fusion Protein ◽  
Prokaryotic Expression ◽  
Recombinant Plasmid ◽  
Nitrilotriacetic Acid ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Pcr Product ◽  
Prokaryotic Expression Vector ◽  
Polymerase Chain

AbstractGP41, a major glycoprotein, identified in the occlusion-derived virions (ODV) of baculoviruses, is required for the egress of nucleocapsids from the nucleus in the pathway of budded virion (BV) synthesis. Using the polymerase chain reaction (PCR), the open reading frame (ORF) ofSpodoptera lituranucleopolyhedrovirus (SpltMNPV)gp41gene was obtained from SpltMNPV genomic DNA. The PCR product was cloned into pMD18-T vector to get the recombinant plasmid (pT-gp41). Thegp41gene was recombinedin vitrowith prokaryotic expression vector pQE30 and transformed intoEscherichia coliM15 [pREP4]. The M15 [pREP4] strain, containinggp41recombinant plasmid, expressed a 37.9 kDa 6×His-tag fusion protein after induction with 1 mmol/l isopropylthio-β-d-galactoside (IPTG). The fusion protein was purified with a nickel-nitrilotriacetic acid (Ni–NTA) resin column and used as the immunogen to raise GP41-specific antibody. Western blotting analysis indicated that the antibody was suitable to be used for further analysis of GP41 protein.

scholarly journals Study of the 49 kDa excretory-secretory protein gene of Trichinella nativa and Trichinella spiralis

2007 ◽  
Vol 44 (3) ◽  
pp. 120-125 ◽  
Author(s):  
B. Zheng ◽  
L. Xiao ◽  
X. Wang ◽  
D. Li ◽  
Y. Lu ◽  
...  
Keyword(s):  
Prokaryotic Expression ◽  
Protein Gene ◽  
Trichinella Spiralis ◽  
Secretory Protein ◽  
Rt Pcr ◽  
Prokaryotic Expression Vector ◽  
Close Relationship ◽  
Bamhi Site ◽  
Relationship Of ◽  
Trichinella Nativa

AbstractTo study the function of the 49 kDa excretory-secretory (ES) protein gene (P49) of Trichinella, the genes was amplified by RT-PCR from RNA of Trichinella spiralis and Trichinella nativa and several Chinese Trichinella isolates of domestic animals, and sequenced after being cloned. The amplified products of these parasites produced bands of about 950 bp. The 97.2 % to 100 % nucleotides identity and 94.3 % to 100 % identity of deduced amino acids among P49 gene of these Trichinella strains showed the close relationship of these parasites. The P49 gene of T. nativa was cloned into the BamHI site of the prokaryotic expression vector pET-30a, and the recombinant vector was expressed. The expressed product was 40.8 kDa in size. In Western blot analysis, the expressed product was reactive to sera of mice infected with T. nativa, T. spiralis and their Chinese geographical strains.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

scholarly journals Specific T-cell Responses to CFP10, an Secreted Antigens of Mycobacterium Tuberculosis Protein, in Chinese HIV Positive Individuals

2013 ◽  
Vol 2 (1) ◽  
pp. 6-12
Author(s):  
Bin Zhang ◽  
Wen-hui Lun ◽  
Xing-wang Li ◽  
Qi Wang ◽  
Jun Cheng ◽  
...  
Keyword(s):  
T Cell ◽  
Recombinant Protein ◽  
Recombinant Proteins ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
T Cell Responses ◽  
Hiv Positive ◽  
E Coli ◽  
Cell Responses ◽  
Prokaryotic Expression Vector

Abstract Objective To construct prokaryotic expression vector of CFP-10 gene, and obtain recombinant protein, and the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses, to set up a method to faciliate to detect potential TB infection in China. Methods CFP-10 was cloned into inducible prokaryotic expression vector pET-32a (+) and transfected into E. coli BL21 (DE3). After IPTG induction, the product were verified with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot hybridization were carried out to verify the antigenicity; the recombinant CFP-10 protein was taken as stimulus to detect specific T cell responses in HIV (+) persons with or without clinical manifestation of TB diseases, and HIV (-) controls with or without TB diseases. Results The CFP-10 recombinant protein exsited in the form of inclusion body and accounted for 94% in total bacterial protein of E. coli and the molecular weight is 31 kD; Western blot confirmed the recombinant proteins had high antigenicity; our in-house ELISpot-IFN-γ assay with recombinant antigen derived from CFP-10 proteins showed significant higher frequencies in TB patients with or without HIV infection than that in the healthy controls and only HIV (+) group. Conclusions The recombinant CFP-10 genes can be expressed successfully in prokaryotic expression system of E. coli and recombinant proteins with high antigenicity were obtained, which will set foundation for further study on their immunogenicity and bioinformatics. Our results proved that it is indeed true that some HIV positive patient have high frequencies of TB specific T cell responses, which maybe a clue to find latent TB infection in this population.

scholarly journals Cloning, expression and purification of outer membrane protein PorA of Neisseria meningitidis serogroup B

2011 ◽  
Vol 5 (12) ◽  
pp. 856-862 ◽  
Author(s):  
Fakhri Haghi ◽  
Shahin Najar Peerayeh ◽  
Seyed Davar Siadat ◽  
Mehran Montajabiniat
Keyword(s):  
Recombinant Protein ◽  
Neisseria Meningitidis ◽  
Outer Membrane ◽  
Prokaryotic Expression ◽  
Membrane Vesicle ◽  
Outer Membrane Proteins ◽  
Expression System ◽  
Sds Page ◽  
Prokaryotic Expression System ◽  
Prokaryotic Expression Vector

Introduction: Neisseria meningitidis is a major causative agent of bacterial septicemia and meningitis in humans. Currently, there are no vaccines to prevent disease caused by strains of N. meningitidis serogroup B. PorA is a major component of the outer membrane of N. meningitidis and functions as a cationic porin. This study aimed to clone and determine the expression of PorA. Methodology: A 1200 bp fragment of porA gene was amplified by PCR from serogroup B N. meningitidis and then cloned into prokaryotic expression vector pET-32a. For expression of recombinant protein, pET32a-porA plasmid was transformed into competent Origami B (DE3) cells. Recombinant protein was overexpressed with isopropythio-beta-D-galctoside (IPTG) and affinity purified by Ni-NTA agarose. SDS-PAGE and western blotting were performed for protein determination and verification. Results: Cloning of porA was confirmed by colony-PCR and enzymatic digestion. In comparison with the corresponding sequences of original genes, the nucleotide sequence homology of the cloned porA gene was 97%. IPTG with a dosage of 1.0 mmol/L could efficiently induce protein expression. SDS-PAGE analysis showed that our constructed prokaryotic expression system pET32a-PorA-Origami efficiently produces a target recombinant protein with a molecular weight of 65 kDa. The recombinant PorA was overexpressed as inclusion bodies and reacted with the serum from a rabbit previously immunized with native outer membrane vesicle. Conclusion: This prokaryotic expression system provides an easy method for producing recombinant PorA and may also be useful for the production of other bacterial outer membrane proteins for vaccine studies.

Construction and expression of a prokaryotic expression vector for the goat sry gene

2019 ◽  
Author(s):  
Xiao Wang ◽  
Guang-Xin E ◽  
Shu-Zhu Cheng ◽  
Wei-Wei Ni ◽  
Yue-Hui Ma ◽  
...  
Keyword(s):  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Expression Vector ◽  
Target Gene ◽  
Rabbit Antiserum ◽  
Rabbit Serum ◽  
Molecular Sexing ◽  
Coding Region ◽  
Sry Gene ◽  
Prokaryotic Expression Vector

Goats are economically important animals in the world, and their sex is an important factor in their economic efficiency. Reconstruction of a goat SRY gene expression vector can lay a foundation for studying the immunogenicity and sex determination of SRY protein at the molecular level. In this study, the coding region of the goat SRY gene was used as the target gene fragment for synthesis and optimization, and the cloning vector was used as a template to amplify the target gene and finally ligated to the expression vector pET-SUMO. The recombinant plasmid was then verified by the double restriction enzyme method and transformed into Escherichia coli (DE3). After the induction of expression by Isopropyl â-D-Thiogalactoside (IPTG), the cells were lysed, and SDS-PAGE electrophoresis was performed to observe the expression of the recombinant protein. Additionally, the immunological activity of the recombinant protein was assessed. The target gene was successfully ligated into the prokaryotic expression vector pET-28a; additionally, the prokaryotic expression plasmid pET-SUMO was successfully constructed, and the SRY antigen protein (42 kDa) was expressed. The titer of the rabbit antiserum (PAI-1608012-1) was more than 1:50000, as measured by ELISA, which demonstrated that the titer and the sensitivity of the rabbit serum reached the expected levels. In this study, the prokaryotic expression vector pET-SUMO was successfully constructed. The recombinant protein has high immunopotency and immunoreactivity, which lays a foundation for the preparation of antibodies and the molecular sexing of goats in the future.

scholarly journals Aromatase and breast cancer: W39R, an inactive protein

2002 ◽  
pp. 583-589 ◽  
Author(s):  
C Nativelle-Serpentini ◽  
S Lambard ◽  
GE Seralini ◽  
P Sourdaine
Keyword(s):  
Breast Cancer ◽  
Recombinant Protein ◽  
In Vitro Study ◽  
Japanese Women ◽  
Expression Vectors ◽  
Kinetic Properties ◽  
Aromatase Activity ◽  
Rt Pcr ◽  
Inactive Protein

BACKGROUND: Aromatase (CYP19) catalyzes the conversion of androgens into estrogens. It is in particular involved in development, reproduction and breast cancer. One of its polymorphisms, W39R localized in the N-terminal region of CYP19, significantly decreases breast cancer risk among Japanese women and was chosen for this study. In this work, we studied the structure-function relationships between W39R polymorphism and CYP19 enzyme activity. OBJECTIVE: To examine the kinetic properties of the mutant W39R recombinant protein in transfected human cells devoid of steroidogenic activity. METHODS: Expression vectors for the wild-type or the mutated R39 aromatase were transiently transfected into E293 human embryonal kidney cells. The conversions of androstenedione to estrone and of testosterone and nortestosterone to 17beta-estradiol were assayed by RIA. Expression of recombinant cDNAs was analyzed by semi-quantitative RT-PCR and immunoblotting. RESULTS: W39R recombinant protein was devoid of aromatase activity whatever the substrate used. This absence of activity was not due to the lack of expression of the recombinant enzyme since the mRNA and protein were detected. CONCLUSION: Our present in vitro study shows that the R39 mutant is unable to synthesize estrogens. This work provides a novel observation, being consistent with the fact that Japanese women with the variant allele (arg) have significantly lower risk of developing a breast tumor.

scholarly journals Identification of Human Hepatocyte Proliferation Related Gene C2orf69

2014 ◽  
Vol 3 (1) ◽  
pp. 1-9
Author(s):  
Ren-wen Zhang ◽  
Yong Qiao ◽  
Xiao-hua Hao ◽  
Hong-min Li ◽  
Hui Ren ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Recombinant Protein ◽  
Western Blot ◽  
Polyclonal Antibody ◽  
Prokaryotic Expression ◽  
Liver Cells ◽  
Hepatocyte Proliferation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Gene Fragment

Abstract Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was amplified by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identified by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and specificity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully amplified, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

scholarly journals Inhibitory Effects of Plant Trypsin Inhibitors Msti-94 and Msti-16 on Therioaphis trifolii (Monell) (Homoptera: Aphididae) in Alfalfa

Insects ◽  
2019 ◽  
Vol 10 (6) ◽  
pp. 154 ◽  
Author(s):  
Hailong Zhao ◽  
Hidayat Ullah ◽  
Mark Richard McNeill ◽  
Guilin Du ◽  
Kun Hao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Recombinant Protein ◽  
Prokaryotic Expression ◽  
Survival Rates ◽  
Trypsin Inhibitors ◽  
Gene Clone ◽  
Mrna Levels ◽  
Biological Functions ◽  
Inhibitory Effects ◽  
Prokaryotic Expression Vector

The spotted alfalfa aphid (Therioaphis trifolii (Monell)) is a known destructive pest that can significantly reduce alfalfa yields. Two differentially up-regulated alfalfa trypsin inhibitors ‘Msti-94’ and ‘Msti-16’ in transcriptome were verified in terms of their mRNA levels using RT-qPCR. The prokaryotic expression vector was constructed and its biological functions, including phenotypic and physiological responses, were verified through feeding spotted alfalfa aphids with active recombinant protein mixed with an artificial diet. Gene clone and gene prokaryotic expression confirmed that Msti-94 had a size of 651 bp, encoded 216 amino acids with a predicted protein weight of 23.5 kDa, and a pI value of 6.91. Similarly, the size of Msti-16 was 612 bp, encoded 203 amino acids, and had a predicted protein weight of 22.2 kDa with a pI value of 9.06. We concluded that both Msti-94 and Msti-16 acted as a stomach poison with survival rates reduced to 21.7% and 18.3%, respectively, as compared to the control, where the survival rate was significantly (p < 0.05) higher (60.0%). Aphid reproduction rates were significantly reduced, after 72 h of feeding, in both the Msti-94 and Msti-16 treatments compared to the controls. A concentration of 800 μg/mL (0.8 mg/mL) of recombinant protein and 5000 μg/mL (5 mg/mL) of recombinant expressing bacteria that inhibits the total protease, which ultimately disrupted the activity of trypsin, chymotrypsin, and aminopeptidase.

scholarly journals Cloning and Expression of HBV Infection Related Novel Gene C12orf49

2013 ◽  
Vol 2 (2) ◽  
pp. 55-59
Author(s):  
Xue Meng ◽  
Yue Sun ◽  
Hong-yan Gu ◽  
Hong-shan Wei ◽  
Xing-wang Li
Keyword(s):  
Recombinant Protein ◽  
Western Blot ◽  
Polyclonal Antibody ◽  
Hepg2 Cells ◽  
Sodium Dodecyl ◽  
Cloning And Expression ◽  
Novel Gene ◽  
Prokaryotic Expression Vector ◽  
Inductive Agent

Abstract Objective To clone, express and purify C12orf49 recombinant protein. To prepare rabbit anti-C12orf49 protein polyclonal antibody in order to further elucidate its biological function. Methods PCR was used to amplify the gene C12orf49 in vitro. pET-32a (+)-C12orf49, the recombinant protein prokaryotic expression vector, was transformed into E. coli. IPTG was used as the inductive agent to obtain C12orf49 recombinant protein, and the recombinant protein was analyzed with sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Specific polyclonal antibody was derived from rabbits that immunized by recombinant protein. ELISA and Western blot were used to test its titer and specificity, respectively. MTT cell proliferation experiment was carried out to observe effect of the protein on proliferation of HepG2 cells. Results The C12orf49 recombinant protein was expressed in a large quantity. Data of ELISA indicated that the titer of polyclonal antibody was higher than 1:1 280 000. And the antibody also had a good specificity, confirmed by Western blot. C12orf49 recombinant protein may had a advanced effect on the proliferation of HepG2 cells. Conclusions Using C12orf49 recombinant protein, we can obtain the polyclonal antibody with great titer and good specificity. Human novel gene C12orf49 encoded protein could promote the proliferation of HepG2 cells.

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